In addition to its well-known roles in regulating metabolic homeostasis, leptin signaling in the brain has also been critically involved in cardiovascular and respiration functions. Although the central mechanisms mediating leptin’s effect on metabolic control have been extensively studied and elucidated, the neural substrates by which leptin exert its effect on cardiovascular and respiratory functions remain incompletely understood. Leptin receptors (LepRs) are expressed distinct hypothalamic nuclei, including the lateral hypothalamic area (LHA), a brain region historically known as the “feeding center”. We recently performed anterograde tract-tracing of LHA LepR-positive neurons and observed a broad but regionally enriched pattern of projections throughout the brain, including, but not limited to the brain regions involved in sympathetic control of cardiovascular function and breathing, such as the periaqueductal gray and ventrolateral medullar. Based on these preliminary neuroanatomical observations, we hypothesized that the LHA leptin circuit is critically involved in cardiorespiratory control. To test this hypothesis, we generated mice expressing hM3Dq-mCherry or mCherry (control) specifically in LHA LepR+ neurons by performing stereotaxic microinjection of Cre-dependent AAV-DIO-hM3Dq-mCherry or AAV-DIO-mCherry into the LHA of LepR-Cre+ mice. These mice were then subjected to radiotelemetry recording of blood pressure and whole-body plethysmography to evaluate the respiratory function. We found that chemogenetic activation of LHA LepR+ neurons by intraperitoneal injection of deschloroclozapine (DCZ, 0.5 mg/Kg) significantly elevated blood pressure (p<0.01) and heart rate (p<0.01) independently of locomotor activity. Whole-body plethysmography further revealed that activation of LHA LepR+ neurons also drastically increased the respiratory rate (p<0.0001) and minute ventilation (p<0.001) without a significant change in the tidal volume. Increased respiratory rate was associated with shortening both inspiratory and expiratory durations. These results identify the LHA as a novel hypothalamic node through which the circulating metabolic hormone leptin might influence cardiorespiratory control. This work is supported by the grants from the National Institute of Health (HL127673 and HL153274 to HC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract Sleep and metabolism are inextricably linked and mutually affect each other. Leptin is a pivotal regulator of metabolic homeostasis, but its effect on sleep-wake regulation remains elusive. Here we demonstrate that leptin acts on a small subset of lateral hypothalamic area (LHA) GABAergic neurons to affect sleep-wake behavior. We found that the selective loss of leptin receptors (LepRs) in the LHA causes sleep fragmentation without altering total sleep time, while severe sleep fragmentation in obese LepR-null mice can be rescued by the selective restoration of LHALepR signaling. In vivo Ca2+ imaging revealed that the vast majority of LHALepR+ neurons are REM sleep- and/or wake-active, and chemogenetic activation of LHALepR+ neurons lead to sustained wakefulness. Furthermore, optogenetic activation of LHALepR+ neuron projections to the ventral tegmental area promotes arousal. Collectively, our results identify an important hypothalamic substrate linking metabolic alterations to aberrant sleep-wake patterns in obesity.

Hippocampal dysfunction is associated with major depressive disorder, a serious mental illness characterized by not only depressed mood but also appetite disturbance and dysregulated body weight. However, the underlying mechanisms by which hippocampal circuits regulate metabolic homeostasis remain incompletely understood. Here we show that collateralizing melanocortin 4 receptor (MC4R) circuits in the ventral subiculum (vSUB), one of the major output structures of the hippocampal formation, affect food motivation and energy balance. Viral-mediated cell type- and projection-specific input-output circuit mapping revealed that the nucleus accumbens shell (NAcSh)-projecting vSUBMC4R+ neurons send extensive collateral projections of to various hypothalamic nuclei known to be important for energy balance, including the arcuate, ventromedial and dorsomedial nuclei, and receive monosynaptic inputs mainly from the ventral CA1 and the anterior paraventricular nucleus of thalamus. Chemogenetic activation of NAcSh-projecting vSUBMC4R+neurons lead to increase in motivation to obtain palatable food without noticeable effect on homeostatic feeding. Viral-mediated restoration of MC4R signaling in the vSUB partially restores obesity in MC4R-null mice without affecting anxiety- and depression-like behaviors. Collectively, these results delineate vSUBMC4R+ circuits to the unprecedented level of precision and identify the vSUBMC4R signaling as a novel regulator of food reward and energy balance.

Norepinephrine (NE) is a well-known appetite regulator, and the nor/adrenergic system is targeted by several anti-obesity drugs. To better understand the circuitry underlying adrenergic appetite control, here we investigated the paraventricular hypothalamic nucleus (PVN), a key brain region that integrates energy signals and receives dense nor/adrenergic input, using a mouse model. We found that PVN NE level increases with signals of energy deficit and decreases with food access. This pattern is recapitulated by the innervating catecholaminergic axon terminals originating from NTS TH -neurons. Optogenetic activation of rostral-NTS TH → PVN projection elicited strong motivation to eat comparable to overnight fasting whereas its inhibition attenuated both fasting-induced & hypoglycemic feeding. We found that NTS TH -axons functionally targeted PVN MC4R -neurons by predominantly inhibiting them, in part, through α1-AR mediated potentiation of GABA release from ARC AgRP presynaptic terminals. Furthermore, glucoprivation suppressed PVN MC4R activity, which was required for hypoglycemic feeding response. These results define an ascending nor/adrenergic circuit, NTS TH → PVN MC4R , that conveys peripheral hunger signals to melanocortin pathway.

ABSTRACT Objective The paraventricular nucleus of hypothalamus (PVN) is an integrative center in the brain orchestrating a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVN MC4R+ ) is undoubtedly involved in feeding regulation, the neuroanatomical organization of PVN MC4R+ pathway and its role in diverse physiological and behavioral regulations have not been fully understood. Here we aimed to better characterize the input-output organization of PVN MC4R+ neurons and further test their potential functional roles beyond feeding. Methods Using a combination of viral tools, we performed a comprehensive mapping of PVN MC4R+ circuits and tested the effects of chemogenetic activation of PVN MC4R+ neurons on thermogenesis, cardiovascular control and other behavioral regulations beyond feeding. Results We found that PVN MC4R+ neurons broadly innervate many different brain regions known to be important not only for feeding but also for neuroendocrine and autonomic control of thermogenesis and cardiovascular function, including but not limited to preoptic area, median eminence, parabrachial nucleus, locus coeruleus, nucleus of solitary tract, ventrolateral medulla and thoracic spinal cord. Contrary to broad efferent projections, PVN MC4R+ neurons receive monosynaptic inputs from limited brain regions, including medial preoptic nucleus, arcuate and dorsomedial hypothalamic nuclei, and supraoptic nucleus. Consistent with broad efferent projections, chemogenetic activation of PVN MC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure and brown adipose tissue thermogenesis. Strikingly, these physiological changes accompanied an unexpected repetitive bedding-removing behavior followed by hypoactivity and resting-like behavior. Conclusions Our results clarify the neuroanatomical organization of PVN MC4R+ circuits and shed new light on the roles of PVN MC4R+ pathways in autonomic control of thermogenesis, cardiovascular function and other behavioral regulations.

Estrogen receptor alpha (ERα)-mediated estrogen signaling play a pivotal role in both reproductive and non-reproductive functions. Transcriptional regulation of ERα gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ERα variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ERα-null mice for functional investigation of the genetic sufficiency, we unexpectedly produced ERα hypomorphic mice with biased downregulation of a previously unappreciated long ERα isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely maintained alive newborns without significant morphological and pathological changes in reproductive system and the disruption of body weight homeostasis, indicating the vital role of this long isoform in female reproductive function. Collectively, our results define a tissue-specifically enriched long ERα isoform and its preferential role in female reproductive function over body weight homeostasis.

Objective: RGS2 is a GTPase activating protein that modulates GPCR-Ga signaling and mice lacking RGS2 globally exhibit metabolic alterations. While RGS2 is known to be broadly expressed throughout the body including the brain, the relative contribution of brain RGS2 to metabolic homeostasis remains unknown. The purpose of this study was to characterize RGS2 expression in the paraventricular nucleus of hypothalamus (PVN) and test its role in metabolic homeostasis.Methods: We used a combination of RNAscope in situ hybridization (ISH), immunohistochemistry, and bioinformatic analyses to characterize the pattern of Rgs2 expression in the PVN. We then created mice lacking Rgs2 either prenatally or postnatally in the PVN and evaluated their metabolic consequences.Results: RNAscope ISH analysis revealed a broad but regionally enriched Rgs2 mRNA expression throughout the mouse brain, with the highest expression being observed in the PVN along with several other brain regions, such as the arcuate nucleus of hypothalamus and the dorsal raphe nucleus. Within the PVN, we found that Rgs2 is specifically enriched in CRH+ endocrine neurons and is further increased by calorie restriction. Functionally, although Sim1-Cre-mediated prenatal deletion of Rgs2 in PVN neurons had no major effects on metabolic homeostasis, AAV-mediated adult deletion of Rgs2 in the PVN led to significantly increased food intake, body weight (both fat and fat-free masses), body length, and blood glucose levels in both male and female mice. Strikingly, we found that prolonged postnatal loss of Rgs2 leads to neuronal cell death in the PVN, while rapid body weight gain in the early phase of viral-mediated PVN Rgs2 deletion is independent of PVN neuronal loss. Conclusions: Our results provide the first evidence to show that PVN Rgs2 expression is not only sensitive to metabolic challenge but also critically required for PVN endocrine neurons to function and maintain metabolic homeostasis.(c) 2022 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Obesity, a metabolic disorder characterized by an excessive accumulation of body fat, continues to be a major economic and health burden to the global populace, as it is the most significant risk factor for heart disease, diabetes, and hypertension. Genome-wide association studies have identified several genes involved in obesity development, including ADCY3, a gene encoding for the enzyme adenylyl cyclase 3 (AC3). AC3 is a critical mediator of the cyclic adenosine monophosphate (cAMP) signaling pathway and is enriched in the primary cilium, a solitary immotile organelle protruding from most mammalian cells, including neurons. The primary cilia act as the cellular antenna to sense and relay changes in the extracellular microenvironment and mounting evidence support a role for the primary cilia as a critical regulator of the energy homeostasis. Patients carrying loss-of-function ADCY3variants are obese and are at increased risk of developing type II diabetes mellitus (T2DM). Consistently, whole-body Adcy3knockout mice also develop obesity; however, the underlying mechanisms by which AC3 affects metabolic homeostasis remain unclear. Here, we examined the impact of AC3 loss within the ventromedial nucleus of hypothalamus (VMH), a brain region critical for coordinated control of energy and glucose homeostasis, on energy metabolism. We generated mice with VMH-specific Adcy3loss (Adcy3SF-1KO mice) by crossing SF-1 Cre+ mice with Adcy3F/F mice and subjected them to metabolic phenotyping to assess energy balance and glucose homeostasis. We observed a sexually dimorphic effect on body weight under standard chow diet; male Adcy3SF-1KO mice displayed normal body weight gain comparable to control littermates, whereas female Adcy3SF-1KO mice gained a significant more weight compared to control littermates (Con 24.34g v KO 30.61g, p < 0.0001). Strikingly, however, both sexes of Adcy3SF-1KOmice exhibited improved glucose tolerance (AUC: M Con 17,826 v KO 15,165, p = 0.02; F Con 15,263 v KO 13,238, p = 0.03) without affecting fed and overnight fasting blood glucose levels. Taken together, these results underscore the importance of AC3-mediated ciliary cAMP signaling in VMH neurons for the regulation of energy balance and glucose homeostasis in a sex-dependent manner.

Central melanocortin pathways, mainly via acting on melanocortin-4 receptor (MC4R), play an important role in both energy homeostasis and sympathetic control of cardiovascular function. The paraventricular nucleus of hypothalamus (PVN) is one of hypothalamic nuclei with highest MC4R expression and is well known for its role in metabolic and autonomic regulations; however, the role of PVN-MC4R neural pathways in this regard remains incompletely understood. In this study, we first tested whether chemogenetic activation of PVN-MC4R neurons affects cardiovascular function. Adeno-associated virus (AAV) expressing excitatory (hM3Dq) Designer Receptors Exclusively Activated by Designer Drugs (DREADD) receptors were directly injected into the PVN of MC4R-Cre knock-in mice that are implanted with radio-telemetry for chronic monitoring of blood pressure. DREADD activation of PVN-MC4R neurons significantly elevated mean arterial pressure and heart rate. Next, we prepared a separate group of mice and subjected them to a battery of tests to gain insight into the role of these neurons in other behavioral and physiological regulations. Whole-body infrared thermal imaging upon DREADD activation of PVN-MC4R neurons revealed significant increase of temperature in near neck region, lower back, and tail. Behavioral monitoring in PhenoTyper cage further showed that DREADD activation of PVN-MC4R neurons leads to hypoactivity, suppressed feeding, and trend toward increased drinking behavior. In order to gain further insight into the neuroanatomical organization of PVN-MC4R neurons, we comprehensively mapped afferent projections of PVN-MC4R neurons by stereotaxically delivering Cre-dependent AAV driving expression of eYFP into the PVN of MC4R-Cre mice, which revealed a broad innervation of PVN-MC4R neurons to the brain regions that are known to be important for metabolic homeostasis and autonomic-cardiovascular control, including but not limited to parabrachial nucleus, nucleus of solitary tract, ventrolateral medulla, and spinal cord. Additionally, using a combination of sophisticated viral tools, we also mapped the brain regions where neurons provide monosynaptic inputs to PVN-MC4R neurons. In addition to the brain regions in which neurons are known to send monosynaptic inputs to PVN-MC4R neurons, such as arcuate nucleus, few discreate presynaptic neurons were also observed in organum vasculosum of the lamina terminalis, bed nucleus of the stria terminalis, supraoptic nucleus (SON) and ventral subiculum (vSub). These results provide an important insight into the neuroanatomical organization and multifaceted role of PVN-MC4R neural pathways in behavioral and physiological regulations.

Central melanocortin pathways, mainly via acting on melanocortin‐4 receptor (MC4R), play an important role in both energy balance and sympathetic traffics affecting cardiovascular function. MC4R deficiency in both humans and rodents causes obesity, insulin resistance and diabetes while maintaining low to normal sympathetic tone and blood pressure, suggesting a critical role of MC4R signaling in metabolic homeostasis and obesity‐associated sympathetic overdrive and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which functions as an integrative center for energy metabolism and autonomic regulation. However, whether and how PVNMC4R pathways contribute to the sympathetic control of cardiovascular function and metabolic homeostasis remain incompletely understood. In present study, we first comprehensively mapped afferent projections of PVNMC4R neurons by stereotaxically delivering Cre‐dependent AAV driving expression of eYFP into the PVN of MC4R‐Cre knock‐in mice, which allow targeted anterograde tract‐tracing of PVNMC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVNMC4R neurons to various brain regions important for autonomic‐cardiovascular control and energy metabolism, including but not limited to nucleus of solitary tractus , dorsal motor nucleus of vagus, rostral ventrolateral medulla (RVLM) and spinal cord. Using a combination of sophisticated viral tools, we further reveal that thoracic spinal cord‐projecting PVNMC4R neurons are exclusively located in the posterolateral PVN and send collateral projections to RVLM. Functionally, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes renal sympathetic nerve activity in anesthetized mice and chemogenetic activation of PVNMC4R neurons elevates blood pressure and decreases food intake in freely moving animals. Additionally, genetic ablation of PVNMC4R neurons in adult mice using Cre‐dependent AAV expressing Casp3 causes hyperphagia and massive weight gain (over 80% increase within 6 weeks). These results provide an important insight into the role of PVNMCR neural circuits in regulating metabolic homeostasis and cardiovascular function. Optogenetic and chemogenetic studies are ongoing to functionally dissect the neural circuits downstream of PVNMC4R neurons.Support or Funding InformationNational Institute of Health (R01), AHA Postdoctoral Grant Award (19POST34450083)

Apart from reproduction, estrogen influences a multitude of processes. Increase in estrogen levels in women is known to promote reward probably mediated via the melanocortin and dopamine systems. Reduced estrogen in post-menopausal women attenuates reward, evoking the need for stimulation with greater rewarding salience. This is reflected in the well-recognized phenomena of difficulty in quitting and increased craving for nicotine in women following the onset of menopause. The present study aims at understanding the role of melanocortin receptors (MC-R) in nicotine-induced reward behavior following ovariectomy in rats. The MC4-R mRNA level was increased in ipsilateral nucleus accumbens (Acb) of the intact rats implanted with electrode in medial forebrain bundle and trained in intracranial self-stimulation (ICSS) paradigm. Additional groups of ICSS trained rats were ovariectomized (OVX) and subjected to reward evaluation. Trained OVX rats revealed a significant increase in threshold frequency and rightward shift in rate frequency curve, suggesting reward deficit behavior. However, pre-administration with nicotine, alpha-melanocyte stimulating hormone (α-MSH) or NDP-MSH (MC4-R agonist) to OVX animals restored the rewarding activity in ICSS protocol; HS014 (MC4-R antagonist) suppressed the lever press activity. Prior treatment with sub-effective doses of α-MSH or NDP-MSH potentiated the reward effect of nicotine, but was attenuated by HS014. Alpha-MSH-immunoreactivity was decreased in the Acb shell, arcuate and paraventricular nucleus of hypothalamus, and ventral bed nucleus of stria terminalis in the OVX rats, while nicotine treatment restored the same. We suggest a role for the endogenous MC system, perhaps acting via MC4-R, in the nicotine-induced reward in OVX rats.

Most of the animal studies using inflammation-induced cognitive change have relied on behavioral testing without objective and biologically solid methods to quantify the severity of cognitive disturbances. We have developed a bispectral EEG (BSEEG) method using a novel algorithm in clinical study. This method effectively differentiates between patients with and without delirium, and predict long-term mortality. In the present study, we aimed to apply our bispectral EEG (BSEEG) method, which can detect patients with delirium, to a mouse model of delirium with systemic inflammation induced by lipopolysaccharides (LPS) injection. We recorded EEG after LPS injection using wildtype early adulthood mice (2-3-month-old) and aged mice (18-19-month-old). Animal EEG recordings were converted for power spectral density to calculate BSEEG score using the similar BSEEG algorithm previously developed for our human study. The BSEEG score was relatively stable and slightly high during the day. Alternatively, the BSEEG score was erratic and low in average during the night. LPS injection increased the BSEEG score dose-dependently and diminished the diurnal changes. The mean BSEEG score increased much more in the aged mice group as dosage increased. Our results suggest that BSEEG method can objectively "quantify" level of neuro-Inflammation induced by systemic inflammation (LPS), and that this BSEEG method can be useful as a model of delirium in mice.

Leptin resistance is a hallmark of obesity with unclear etiology. Celastrol, a compound found in the roots of the Tripterygium wilfordii and known to reduce endoplasmic reticulum (ER) stress, has recently emerged as a promising candidate to treat obesity by improving leptin sensitivity. However, the underlying neural mechanisms by which celastrol reduces obesity remain unclear. Using three different mouse models of obesity—diet-induced obesity (DIO), leptin receptor (LepR)-null, and melanocortin 4 receptor (MC4R)-null mice—in this study, we show that systemic celastrol administration substantially reduces food intake and body weight in MC4R-null comparable to DIO, proving the MC4R-independent antiobesity effect of celastrol. Body weight reduction was due to decreases in both fat and lean mass, and modest but significant body weight reduction was also observed in nonobese wild-type and LepR-null mice. Unexpectedly, celastrol upregulated proinflammatory cytokines without affecting genes involved in ER stress. Importantly, celastrol steadily increased sympathetic nerve activity to the brown fat and kidney with concordant increases of resting metabolic rate and arterial pressure. Our results suggest a previously unappreciated mechanism of action of celastrol in the regulation of energy homeostasis and highlight the need for careful consideration of its development as a safe antiobesity medication.

Obesity is commonly associated with sympathetic overactivity and elevated blood pressure, and accumulating evidence suggests that an adipocyte-derived metabolic hormone, leptin, may plays an important role in these pathological processes, likely through a mechanism of so-called ‘selective leptin resistance’. However, underlying neural circuits through which leptin causes sympathoexcitation and increases blood pressure remain incompletely understood. Here we hypothesized that leptin may acts on a subset of lateral hypothalamic area (LHA) neurons to affect cardiovascular functions. We show that stereotaxic microinfusion of leptin into the LHA dose-dependently increases renal sympathetic nerve activity (RSNA) (% changes from baseline at 4 th hour: vehicle -25.03 ± 7.09 % vs leptin 100.23 ± 26.94 %, p<0.001) and selective chemogenetic activation of LHA LepR-expressing neurons increase arterial pressure compared to control mice (19.43 ± 0.87 mm of Hg, p<0.05). Consistent with these functional observations, viral-mediated cell type-specific anterograde tracing revealed that LHA LepR-expressing neurons, which are distinct from well-known orexin and MCH neurons, broadly innervate brain regions known for autonomic regulation, including but not limited to paraventricular nucleus, nucleus of solitary tractus, locus coeruleus, and ventrolateral medulla. These findings identify the LHA as a novel brain site critical for leptin to regulate RSNA and arterial pressure.

Thermosensitive transient receptor potential vanilloid (TRPV) channels are widely expressed in the brain and known to profoundly influence Ca2+-signaling, neurotransmitter release and behavior. While these channels are expressed in the cerebellum, neuronal firing and hyperactivity/reflexes seem associated with cerebellar temperature modulation. However, the distribution and functional significance of TRPV-equipped elements in the cerebellum has remained unexplored. Among TRPV sub-family, TRPV3 is regulated by temperature within physiological range and its transcript highly expressed in the brain. The study aims at exploring the relevance of TRPV3 in the cerebellum of developing and adult rat. RT-PCR analysis showed expression of N- and C-terminal fragments of TRPV3 mRNA in the adult rat cerebellum. Using double immunofluorescence, TRPV3-immunoreactivity was observed in Calbindin D28K-labeled Purkinje neurons. The sections of cerebellum from the postnatal rats (P4, P8, P16 and P42) were processed for TRPV3-immunofluorescence. Compared to P4 and P8, the percent fluorescent area of TRPV3-immunoreactivity significantly increased in the cerebellum of P16 and P42 rats. With a view to test the significance of TRPV3 in cerebellar function, TRPV3-agonist (eugenol) or inhibitors [ruthenium red or isopentenyl pyrophosphate (IPP)] were administered stereotaxically intracerebellum and motor responses analyzed. Compared to controls, rats injected with TRPV3 inhibitor significantly reduced the stride length (P < 0.001), locomotor activity (P < 0.001), and rotarod retention time (P < 0.001), but increased footprints length (P < 0.01) and escape latency (P < 0001). TRPV3-agonist treatment, however, had no effect on these behaviors. We suggest that TRPV3 in Purkinje neurons may serve as novel molecular component for Ca2+-signaling and motor coordination function of the cerebellum. (C) 2019 IBRO. Published by Elsevier Ltd. All rights reserved.

It is well established that the central melanocortin system is critical for autonomic functions and energy homeostasis mainly via signaling at melanocortin-4 receptor (MC4R). Importantly, while obesity is commonly associated with elevated sympathetic tone and blood pressure, severely obese humans and rodents due to genetic MC4R deficiency exhibit normal to low sympathetic tone and blood pressure, suggesting a significant role of MC4R pathway in mediating obesity-associated sympathoexcitation and hypertension. MC4R is widely expressed in the brain including hypothalamic paraventricular nucleus (PVN) which regulates feeding and sympathetic traffic. However, the neuroanatomical basis of PVN MC4R neurons for sympathetic regulation is unclear. The goal of current study is to map the PVN MC4R neural circuits affecting sympathetic tone in mice. To this end, we injected Cre-dependent AAV driving eYFP-fused channel rhodopsin-2 expression into the PVN of MC4R-t2a-Cre knock-in mice, which allow targeted anterograde tract-tracing of PVN MC4R neurons throughout the brain. In addition to known brain region for feeding behavior (i.e. parabrachial nucleus), we found broad innervations of PVN MC4R neurons to various brain regions important for autonomic-cardiovascular control, including, but not limited to, nucleus of solitary tractus, dorsomotor nucleus of vagus, and ventrolateral medulla. Considerable innervation was also evident in spinal cord, which is further confirmed by Fluoro-gold (FG)-mediated retrograde tracing in the spinal cord (thoracic T6-10) of MC4R-GFP transgenic mouse. Double immunofluorescence labeling of GFP and FG revealed that ~50% (58 out of 116) MC4R neurons in the posterior parvocellular subdivision of PVN project to thoracic spinal cord. Furthermore, microinjection of synthetic MC4R agonist (MTII) into the PVN evokes ~42% increase (from baseline) in renal sympathetic nerve activity in anesthetized mice. These results provide important insights into understanding the divergent neural circuits by which PVN MC4R signaling differentially regulates metabolic and cardiovascular functions. Functional dissection of these diverging neural pathways using optogenetic/chemogenetic approaches is ongoing.

In recent years estradiol has emerged as a potential regulator of transient receptor potential vanilloid (TRPV) cationic channels in the peripheral tissues and sensory neurons, however, its analogous role in the CNS is poorly understood. TRPV channels modulate Ca2+ signalling, neurotransmission and behaviour, and expression of these ion channels and estrogen receptors show a great degree of overlap in different brain regions. Herein, we probe if Trpv1-6 genes contain estrogen receptor-binding sites and if their expression in different brain regions is modulated during estrous cycle. Bioinformatics analysis of the mouse Trpv1-6 gene sequences showed presence of putative functional estrogen response element in their promoter regions. Using qRT-PCR, Trpv1-6 mRNA expression was observed in the olfactory bulb, cortex, hypothalamus, hippocampus, brainstem, and cerebellum of mouse. In these regions, compared to estrus, metestrus, and diestrus, reduced levels of Trpv1 and Trpv5 but elevated Trpv2 and Trpv6 mRNA levels were observed during proestrus. Lower levels of Trpv3 and Trpv4 mRNAs were seen during estrus but higher expression of Trpv3 during metestrus and diestrus, and Trpv4 during proestrus was observed. Estradiol seems to regulate Trpv1/Trpv5 and Trpv2/Trpv6 mRNA expression in opposite manner. Except Trpv4 mRNA expression in the hippocampus and Trpv6 expression in the olfactory bulb, hippocampus and brainstem, expression of other members of TRPV subfamily in distinct brain regions of male mice was comparable to those in metestrus and diestrus mice. We suggest that the circulating levels of estradiol during the estrous cycle may differentially regulate the activity of TRPV1-6 ion channels in the brain.

Recent studies suggest an important role for transient receptor potential vanilloid ( TRPV ) ion channels in neural and neuroendocrine regulation. The TRPV subfamily consists of six members: TRPV 1‐6. While the neuroanatomical and functional correlates of TRPV 1‐4 have been studied extensively, relevant information about TRPV 5 and TRPV 6, which are highly selective for Ca 2+ , is limited. We detected TRPV 5 mRNA expression in the olfactory bulb, cortex, hypothalamus, hippocampus, midbrain, brainstem and cerebellum of the rat. TRPV 5‐immunoreactive neurones were conspicuously seen in the hypothalamic paraventricular ( PVN ), supraoptic ( SON ), accessory neurosecretory ( ANS ), supraoptic nucleus, retrochiasmatic part (SOR), arcuate ( ARC ) and medial tuberal nuclei, hippocampus, midbrain, brainstem and cerebellum. Glial cells also showed TRPV 5‐immunoreactivity. To test the neuroendocrine relevance of TRPV 5, we focused on vasopressin, oxytocin and cocaine‐ and amphetamine‐regulated transcript ( CART ) as representative candidate markers with which TRPV 5 may co‐exist. In the hypothalamic neurones, co‐expression of TRPV 5 was observed with vasopressin ( PVN : 50.73±3.82%; SON : 75.91±2.34%; ANS : 49.12±4.28%; SOR : 100%) and oxytocin ( PVN : 6.88±1.21; SON : 63.34±5.69%; ANS : 20.4±4.14; SOR : 86.5±1.74%). While ARC neurones express oestrogen receptors, 17β‐oestradiol regulates TRPV 5, as well as CART neurones and astrocytes, in the ARC . Furthermore, ARC CART neurones are known to project to the preoptic area, and innervate and regulate Gn RH neurones. Using double‐immunofluorescence, glial fibrillary acidic protein ‐labelled astrocytes and the majority of CART neurones in the ARC showed TRPV 5‐immunoreactivity. Following iontophoresis of retrograde neuronal tracer, cholera toxin β (CtB) into the anteroventral periventricular nucleus and median preoptic nucleus, retrograde accumulation of CtB was observed in most TRPV 5‐equipped ARC CART neurones. Next, we determined the response of TRPV 5‐elements in the ARC during the oestrous cycle. Compared to pro‐oestrus, a significant increase ( P <.001) in the percentage of TRPV 5‐expressing CART neurones was observed during oestrus, metoestrus, and dioestrus. TRPV 5‐immunoreactivity in the astrocytes, however, showed a significant increase during metoestrus and dioestrus. We suggest that the TRPV 5 ion channel may serve as an important regulator of neural and neuroendocrine pathways in the brain.

While dopamine (DA) neurons in the ventral tegmental area (VTA) drive the mesolimbic-reward pathway, confluent lines of evidence underscore the importance of transient receptor potential vanilloid (TRPV) channels as novel regulators of these neurons. Among the TRPV-subfamily, TRPV3 is of particular interest in reward, since active ingredients of flavour-enhancing spices in food serve as TRPV3 agonists and modulate DAergic neurotransmission. The nature of TRPV3 elements in the VTA and their role in driving the mesolimbic-DA-reward pathway has however, remained unexplored. We observed TRPV3 mRNA as well as TRPV3-immunoreactive neurons in the VTA of Wistar rats. We therefore explored whether these ion channels participate in modulating mesolimbic-DA reward pathway. In the posterior VTA (pVTA), 82 ± 2.6% of the TRPV3 neurons co-express tyrosine hydroxylase and 68 ± 5.5% of these neurons project to the nucleus accumbens shell (Acb shell). While ex vivo treatment of midbrain slices with TRPV3-agonist, thymol increased [Ca(2+)]i-activity in pVTA neurons, intra-pVTA injections of thymol in freely-moving, satiated rats enhanced positive reinforcement for active lever pressings in an operant chamber to self-administer sweet pellets. This behavior was attenuated by prior treatment with intra-Acb shell DA D1- and D2-like receptor antagonists. These results demonstrate a role for TRPV3 in driving mesolimbic-DA food-reward pathway, and underscores the importance of these channels in the VTA as key components processing reward.